SynopsisMeasurements of the relative quantum yield of fluorescence of proflavine bound to DNA as a function of the number of bound dyes per nucleotide and the ionic strength allow the determination of the binding constants and respective number of the two types of sites previously postulated. It is demonstrated that 2-3% of the base pairs form sites where the dye is strongly bound and fluoresces normally while in the other set of sites the binding constant is 3-4 times weaker and the fluorescence completely quenched.Comparison with complexes of Pro with double stranded polynucleotides poly (A + U), poly (I + C), poly (G + C), confirm that the strong binding sites correspond to A-T-rich regions of the I>XA while the quenched sites seem to require the presence of a neighboring guanine. The role of charge transfer in quenching of fluorescence and mutagnic act,ion is considered. An original method for the determination of free dye and bound dye, based upon the use of an external quencher is described in the Appendix.
Methionyl-tRNA Synthetase from Wheat Embryo : Dissociation into SubunitsWheat-embryo methionyl-tRNA synthetase is a dimeric protein of PZ structure. When highly diluted, it loses the capacity to catalyze ATP-[32P]PPi exchange and to aminoacylate tRNAM": at low enzymatic concentrations the rates of formation of r2P]ATP and [14C]methionyl-tRNAMet are lower than those predicted by extrapolating the rates determined at higher enzyme concentrations. The difference between observed and expected rates becomes greater with decreasing enzyme concentration.Filtration of purified, dilute enzyme preparations on Sephadex G-200 results in the separation of dimer and monomer fractions. The proportion of monomer present increases with increasing pre-incubation times before the assay and demonstrates an equilibrium between active dimers and inactive monomers. The above-mentioned loss of enzymatic activity is explained by the equilibrium being shifted towards the production of monomers. Data previously gathered for Escherichia coli prolyl-tRNA synthetase and for bovine-pancreatic tryptophanyl-tRNA synthetase, coupled with the present results, suggests that the dissociation of dimeric synthetases may be a general phenomenon in eukaryotes as well as in prokaryotes.The number of sub-units and the dissociation constant were obtained at equilibrium, according to relations adapted to the case of oligomeric enzymes (KD = 13 nM at 25 "C and pH 7.5).Rate constants were determined by kinetic studies of the attainment of equilibrium. The rate constant kl for monomolecular dissociation was determined to be 1.85 .s-' and k~ for the bimolecular association to be 0.145 . lo6 M-' s-' . The KD calculated from kl and kz was coherent with the experimentally determined value, at equilibrium.The sub-unit interactions, which involve only a small quantity of energy (AGO = + 11 kcal mol-'; +45 kJ mol-') at 25 "C, depend on the ionic environment of the medium and the presence of substrates. Alkaline pH favors monomer production, while the presence of methionine, (Mg-ATP)2 -and tRNAMe' protect the synthetase from dissociation. 2-mercaptoethanol and dithioerythritol prevent only slightly the loss of activity. Bovine serum albumin, however, protects the enzyme from dissociation under dilute conditions.Dans les reactions catalyskes par de nombreuses enzymes, les vitesses initiales de formation des produits sont proportionnelles aux concentrations enzymatiques utilisees. Ce n'est pas le cas de la methionyltRNA synthetase des embryons de blk, proteine de poids molkculaire 165000 et de structure dimereAbreviations. Unite ,4260, quantite de materiel contenue dans 1 ml d'une solution qui a une absorbance de 1 a 260 nm, pour un trajet optique de 1 cm.Enzyme. Methionyl-tRNA synthetase ou L-mtthionine : tRNA ligase (EC 6.1.1.10). P 2 [l]. Cette derniere perd, aux faibles concentrations enzymatiques, son aptitude a catalyser l'echange ATP-[32P]PPi ainsi que l'aminoacylation des tRNAMe'. Dans ce present travail, nous montrons que l'equilibre entre dimeres actifs et mo...
ABSTRACT-2-Thiazoline-2-thiol is an antithyroid agent that strongly reduces thyroid hormone levels. Synthesis of these hormones is catalyzed in vivo by thyroid peroxidase. The interaction of this drug with molecular iodine and its effect on peroxidase activity were investigated. Iodine and 2-thiazoline-2-thiol form a complex of the charge transfer type of 1:1 stoichiometry characterized by a formation constant of 2,5271-mole-1 at 20°C. This drug was found to inhibit both horseradish peroxidase and lactoperoxidase (used as a model of thyroid peroxidase) in a competitive manner, giving inhibition con stants of 5.7 mM and 0.13 mM, respectively. T3 and T4 levels were reduced significantly after a three-week administration of this drug to a group of 10 rats. Histological ex amination of the thyroid gland showed the presence of a cylindrical epithelium, which is indicative of hyperactivity of the gland. The results indicated that 2-thiazoline-2-thiol acts on both molecular iodine and thyroid peroxidase.Synthetic antithyroid agents are thought to exert their action on thyroid peroxidase, the enzyme responsible for the oxidation of iodides and the coupling of iodotyrosines to iodothyronines (1, 2). We showed that they may also interact with molecular iodine (3). Iodine is complexed by the drug, and thus be comes unavailable for synthesis of thyroid hor mones. A drug which has activity towards TPO and which can complex molecular iodine will thus have particularly strong antithyroid activity. In the present study, we investigated the action of the antithyroid drug 2T2T (4), which has a partially hydrogenated thiazole nucleus. This drug also contains a thiol group, which is also found in MMI, a powerful anti thyroid agent. We also tested action of 2T2T in vivo in the rat to find out whether the com bined actions on peroxidase and iodine did in fact confer strong antithyroid activity.
1. The Michaelis constants in the tRNA aminoacylation reaction have been studied for the three dimeric glutamyl-tRNA synthetases C, P and E. The values were found to be: for tRNA, 0.20 pM, 0.20 pM and 0.44 p M ; for glutamic acid, 10 pM, 83 pM and 83 p M ; for MgATP, 0.46 mM, 0.38 m M and 0.26 mM. MgATP concentrations higher than 2 mM induce pronounced inhibition.2. The presence of the cognate tRNA is required for ["P]PP,-ATP isotopic exchange. In the absence of tRNA no hyperbolic saturation of the enzymes by glutamic acid occurs in our experimental conditions.3 . Analysis of the enzymic activity as a function of enzyme concentration leads to the conclusion that the active forms are dimers which are in equilibrium with inactive monomers. The values of the dissociation constants Kd were found to be 43 nM, 53 n M and 87 nM for glutamyl-tRNA synthetases C, P and E respectively.We have reported in the preceding paper [I] the purification of three wheat dimeric glutamyl-tRNA synthetases (GluRSs). GluRS C, isolated from chloroplasts, exhibits a molecular weight of I10000 and a prokaryotic tRNA specificity. In the quiescent wheat germ two GluRSs, called P and E, were found : both exhibit identical apparent molecular weight, approximately 160000; the Pform, withprokaryotic tRNA specificity, may be of mitochondria1 origin; the E form exhibits eukaryotic tRNA specificity.In the present study the catalytic properties of the three enzymes were compared in the t RNA aminoacylation reaction and in the PPi-ATP exchange reaction. The results allowcd us to define optimal assay conditions. Because enzymic activity was found to vary with enzyme concentration, as observed for wheat inethionyl-t RNA [2] and leucyl-tRNA [3] synthetases, dilution experiments wcre carried out in order to look for thc functional enzymatic structures. MATERIALS AND MhTHODS EnzymesThe three wheat dimeric GluRSs, C, P and E, were obtained Cliernicu1.s[32P]PP, (81 Ci/mol) was purchased from Commissariat ; I I'Energie Atomique (Saclay, France). Unlabeled sodium pyrophosphate was from Merck (Darmstadt, FRG). Charcoal Norite A was from Prolabo (France). The other chemicals have been described in the preceding paper [l].Ahhrrviation. GIuRS, glutamyl-t KNA synthetase. Enzyme. Glutamyl-tRNA synthctase (EC 6.1.1.17). Enzyme assaysThe t RNA aminoacylation reaction was performed according to Ratinaud et al. [I] for usual assays. For activity analysis substrate saturation conditions, except for ATP, were used. ( 3 2 P ] P P i -A T P isotopic e.whungc assayThis activity assay was performed by a technique similar to that described by Cole and Schimmel[5], using 0.1-ml mixtures containing HCl/imidazole buffer (0.1 M) pH 7.5, MgCI, ( 5 m M or 10mM), NaATP (2mM), [32P]PPi ( 2 m M , XI Ci/mol), glutarnic acid (25 rnM or 100 mM), tRNAG'" (various concentrations) and enzyme in amounts so that initial rate could be obscrved. Incubation was carried out at 25 C for 15 min. The reaction was terminated by addition of 2.5 ml of a 77; perchloric acid containing 0.2 M unlabeled so...
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